Jerry Leung, Asel Primbetova, Colton Strong, Brenna N Hay, Han Hsuan Hsu, Andrew Hagner, Leonard J Foster, Dana Devine, Pieter R Cullis, Peter W Zandstra, Christian J Kastrup
{"title":"利用 mRNA 脂质纳米颗粒对血液祖细胞中的巨核细胞进行基因工程改造。","authors":"Jerry Leung, Asel Primbetova, Colton Strong, Brenna N Hay, Han Hsuan Hsu, Andrew Hagner, Leonard J Foster, Dana Devine, Pieter R Cullis, Peter W Zandstra, Christian J Kastrup","doi":"10.1016/j.jtha.2024.09.008","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Platelets are an essential component of hemorrhage control and management, and engineering platelets to express therapeutic proteins could expand their use as a cell therapy. Genetically engineered platelets can be achieved by modifying the platelet precursor cells, megakaryocytes (MKs). Current strategies include transfecting MK progenitors ex vivo with viral vectors harboring lineage-driven transgenes and inducing the production of in vitro modified platelets. The use of viruses, however, poses challenges in clinical implementation, and no methods currently exist to genetically modify MKs with nonviral techniques. Lipid nanoparticles (LNPs) are a nonviral delivery system that could enable a facile strategy to modify MKs with a variety of nucleic acid payloads.</p><p><strong>Objectives: </strong>To investigate whether LNPs can transfect cultured hematopoietic stem/progenitor cell-derived MKs to express exogenous proteins and induce functional changes.</p><p><strong>Methods: </strong>MK and MK progenitors differentiated from cord blood-derived hematopoietic stem/progenitor cells were treated with LNP formulations containing messenger RNA and resembling the clinically approved LNP formulations. Transfection efficiency was assessed through flow cytometry by expression of enhanced green fluorescent protein. Functional changes to the MKs were assessed through rotational thromboelastometry by expression of exogenous coagulation factor (F)VII, a representative physiologically relevant protein.</p><p><strong>Results: </strong>LNPs enabled transfection efficiencies of 99% in MKs and did not impair MK maturation, viability, and morphology. MKs engineered to express exogenous FVII decreased clotting time in FVII-deficient plasma following clot initiation.</p><p><strong>Conclusion: </strong>This approach provides an easy-to-use modular platform to genetically modify MK and MK progenitors, which can be potentially extended to producing genetically modified cultured platelets.</p>","PeriodicalId":17326,"journal":{"name":"Journal of Thrombosis and Haemostasis","volume":" ","pages":""},"PeriodicalIF":5.5000,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Genetic engineering of megakaryocytes from blood progenitor cells using messenger RNA lipid nanoparticles.\",\"authors\":\"Jerry Leung, Asel Primbetova, Colton Strong, Brenna N Hay, Han Hsuan Hsu, Andrew Hagner, Leonard J Foster, Dana Devine, Pieter R Cullis, Peter W Zandstra, Christian J Kastrup\",\"doi\":\"10.1016/j.jtha.2024.09.008\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>Platelets are an essential component of hemorrhage control and management, and engineering platelets to express therapeutic proteins could expand their use as a cell therapy. Genetically engineered platelets can be achieved by modifying the platelet precursor cells, megakaryocytes (MKs). Current strategies include transfecting MK progenitors ex vivo with viral vectors harboring lineage-driven transgenes and inducing the production of in vitro modified platelets. The use of viruses, however, poses challenges in clinical implementation, and no methods currently exist to genetically modify MKs with nonviral techniques. Lipid nanoparticles (LNPs) are a nonviral delivery system that could enable a facile strategy to modify MKs with a variety of nucleic acid payloads.</p><p><strong>Objectives: </strong>To investigate whether LNPs can transfect cultured hematopoietic stem/progenitor cell-derived MKs to express exogenous proteins and induce functional changes.</p><p><strong>Methods: </strong>MK and MK progenitors differentiated from cord blood-derived hematopoietic stem/progenitor cells were treated with LNP formulations containing messenger RNA and resembling the clinically approved LNP formulations. Transfection efficiency was assessed through flow cytometry by expression of enhanced green fluorescent protein. Functional changes to the MKs were assessed through rotational thromboelastometry by expression of exogenous coagulation factor (F)VII, a representative physiologically relevant protein.</p><p><strong>Results: </strong>LNPs enabled transfection efficiencies of 99% in MKs and did not impair MK maturation, viability, and morphology. MKs engineered to express exogenous FVII decreased clotting time in FVII-deficient plasma following clot initiation.</p><p><strong>Conclusion: </strong>This approach provides an easy-to-use modular platform to genetically modify MK and MK progenitors, which can be potentially extended to producing genetically modified cultured platelets.</p>\",\"PeriodicalId\":17326,\"journal\":{\"name\":\"Journal of Thrombosis and Haemostasis\",\"volume\":\" \",\"pages\":\"\"},\"PeriodicalIF\":5.5000,\"publicationDate\":\"2024-09-26\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Thrombosis and Haemostasis\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1016/j.jtha.2024.09.008\",\"RegionNum\":2,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"HEMATOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Thrombosis and Haemostasis","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1016/j.jtha.2024.09.008","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"HEMATOLOGY","Score":null,"Total":0}
Genetic engineering of megakaryocytes from blood progenitor cells using messenger RNA lipid nanoparticles.
Background: Platelets are an essential component of hemorrhage control and management, and engineering platelets to express therapeutic proteins could expand their use as a cell therapy. Genetically engineered platelets can be achieved by modifying the platelet precursor cells, megakaryocytes (MKs). Current strategies include transfecting MK progenitors ex vivo with viral vectors harboring lineage-driven transgenes and inducing the production of in vitro modified platelets. The use of viruses, however, poses challenges in clinical implementation, and no methods currently exist to genetically modify MKs with nonviral techniques. Lipid nanoparticles (LNPs) are a nonviral delivery system that could enable a facile strategy to modify MKs with a variety of nucleic acid payloads.
Objectives: To investigate whether LNPs can transfect cultured hematopoietic stem/progenitor cell-derived MKs to express exogenous proteins and induce functional changes.
Methods: MK and MK progenitors differentiated from cord blood-derived hematopoietic stem/progenitor cells were treated with LNP formulations containing messenger RNA and resembling the clinically approved LNP formulations. Transfection efficiency was assessed through flow cytometry by expression of enhanced green fluorescent protein. Functional changes to the MKs were assessed through rotational thromboelastometry by expression of exogenous coagulation factor (F)VII, a representative physiologically relevant protein.
Results: LNPs enabled transfection efficiencies of 99% in MKs and did not impair MK maturation, viability, and morphology. MKs engineered to express exogenous FVII decreased clotting time in FVII-deficient plasma following clot initiation.
Conclusion: This approach provides an easy-to-use modular platform to genetically modify MK and MK progenitors, which can be potentially extended to producing genetically modified cultured platelets.
期刊介绍:
The Journal of Thrombosis and Haemostasis (JTH) serves as the official journal of the International Society on Thrombosis and Haemostasis. It is dedicated to advancing science related to thrombosis, bleeding disorders, and vascular biology through the dissemination and exchange of information and ideas within the global research community.
Types of Publications:
The journal publishes a variety of content, including:
Original research reports
State-of-the-art reviews
Brief reports
Case reports
Invited commentaries on publications in the Journal
Forum articles
Correspondence
Announcements
Scope of Contributions:
Editors invite contributions from both fundamental and clinical domains. These include:
Basic manuscripts on blood coagulation and fibrinolysis
Studies on proteins and reactions related to thrombosis and haemostasis
Research on blood platelets and their interactions with other biological systems, such as the vessel wall, blood cells, and invading organisms
Clinical manuscripts covering various topics including venous thrombosis, arterial disease, hemophilia, bleeding disorders, and platelet diseases
Clinical manuscripts may encompass etiology, diagnostics, prognosis, prevention, and treatment strategies.